Liposomes are now well recognized as useful for delivery of therapeutic agents, such as cytotoxic drugs or other macromolecules capable of modifying cell behaviour, to in vivo sites. For example, U.S. Pat. No. 3,993,754, inventors Rahman, et al, issued Nov. 23, 1976, discloses an improved method for chemotherapy of malignant tumors in which an antitumor drug is encapsulated within liposomes and the liposomes containing the encapsulated drug are injected into an animal or man.
It has been suggested that target, or in vivo site, specificity might be conferred on liposomes by their association with specific antibodies or lectins. Methods of associating antibodies with liposomes have been described, and may be generally divided into two groups--nonspecific association and covalent attachment.
Nonspecific association appears to rely upon the affinity of the Fc portion of the antibody for the hydrophobic region of the lipid bilayer. However, nonspecific association appears incapable of associating more than about 15-30 microgram per micromole of lipid. Also, nonspecific association has little practical value because the liposomes are rendered more permeable than their encapsulated contents and protein aggregation is produced during formation of the nonspecifically associated liposome-protein.
Prior to preparation of the covalently attached protein of coupled-protein species described in U.S. patent application Ser. No. 129,654, attempts to covalently attach protein to liposomes had been unsatisfactory. For example, some of the prior attempts had involved modifications of the proteins which tended to denature the protein, and thus a substantial loss of biological activity had ensued. Other attempts to covalently attach protein to liposomes had produced very small amounts of specific attachment.
By contrast, activated liposomes in accordance with U.S. patent application Ser. No. 129,654 are readily and efficiently covalently bound to a variety of biologically active proteins with at least about 40 microgram of protein per micromole of lipid. For example, use of the activated liposomes has achieved coupling of up to about 200 microgram of F(ab').sub.2 per micromole of lipid; further, such coupled liposome-protein species were shown to have an improved hemagglutinating titre by comparison to the original, soluble antibody from which they were derived.
Very recently, another efficient method for coupling protein to liposomes has reported coupling of up to 600 microgram of Fab' per micromole of phospholipid via a disulfide interchange reaction. Martin, et al., Biochemistry, 20, pages 4429-4238 (July, 1981).
Meanwhile, agglutination methods are known and useful for applications such as blood typing. However, many such methods have had to be performed indirectly, or have been of relatively low sensitivity. For example, the Coombs test is an indirect agglutination method in the sense that a secondary, or intermediate, antibody must be used. Further, detection of antibodies which do not produce positive agglutination (e.g. visible agglutination) when combined with their specific antigens has presented difficulties in applications such as blood crossmatching. Such serologically "incomplete" antibodies are believed to be fully functional bivalent IgG molecules, but they are unable to bridge two cells and hence do not produce positive agglutination.